The nuclear pore complex (NPC) is the only known mediator of nucleocytoplasmic transport across the nuclear envelope (NE). At ~50 MDa it is one of the largest assemblies of proteins in eukaryotic cells. For proper function, the NPC must assemble precisely, establish and maintain correct NE curvature at its anchor site, and selectively transport diverse types of macromolecules. To understand how the NPC performs each of these functions will require high-resolution structures of the NPC and its complexes. I propose to generate high-resolution structures for two NPC sub-complexes (the Nup84 and Nup170 complexes) from S. cerevisiae. Together, these complexes form the core scaffold of the NPC which represents ~50% of its total mass. Each complex will be reconstituted in E. coli using polycistronic expression. Purified complexes will then be analyzed by negative stain and (ultimately) cryo-electron microscopy (EM) to determine the overall topology of each complex. An immunolocalization approach will be taken such that the relative position of each protein in the entire complex will be mapped. This will allow for the assignment of spatial positions for each protein in relation to all others in the complex. Crosslinking studies, combined with mass spectrometry, will be used to identify particular amino acids which participate in protein-protein interactions as well as those amino acids which lie at the surface of complexes. In combination with previously determined fold assignments for each protein, this spatial information will describe which protein folds interact to form an intact complex. As morphology data and crosslinking data provide different types of spatial information, the information from these two methodologies act synergistically. To take full advantage of both data sets, each will be converted into spatial restraints and inputted into the MODELLER software program such that high-precision maps of each complex will be produced. LAY SUMMARY: Our genetic information, DNA, is contained within a specific compartment in each of our cells called the nucleus. For cells to survive, precisely regulated instructions must be sent to and from our DNA in the nucleus to the rest of the cell. Defects in this process can result in diseases, such as cancer. Here, I will examine the structure of the "portal" through which instructions are passed using a combination of structural techniques. This information can then be used to generate detailed maps of the portal. This will enable a new level of understanding about how specific instructions are sent into and out of the nucleus. [unreadable] [unreadable] [unreadable]